The present invention relates to in vitro procedures for DNA replication, and more particularly provides a system for initiating semi-conservative DNA replication under somatic cell cycle control. It also relates to induction of premature competence to replicate. It also relates to the use of such a system, for example in identifying agents that modulate DNA replication, in particular inhibit or stimulate it, thereby providing for example agents having utility based on therapeutic potential.
The mechanisms which regulate the eukaryotic cell cycle are complex and the subject of much research. There is a continuing need for the development of good model systems which allow investigation of the mechanisms of action of particular components of the cell cycle under controlled conditions to provide insights into the control of DNA replication and its coupling to the cell cycle in eukaryotic, particularly human, cells. In addition, such systems will allow a range of uses in deriving products of practical benefit, such as screening and identifying therapeutic agents which inhibit DNA replication, and which could thus be used as anti-cancer drugs, and also agents which stimulate DNA replication, which could be used for tissue repair therapeutics.
An important aspect of a good model system is one in which a population of cells, or cellular nuclei, are synchronized with respect to the cell cycle. In proliferating cells, the cell cycle can be divided into four main stages. Following the production of a new cell by mitotic division, there is a period of time, G1, prior to the start of DNA synthesis in the S phase. During the S phase the genome of the cell is replicated and this is followed by an interval, G2, prior to mitosis (the M phase). Following mitosis, the cells reenter the G1 phase. Non-replicating cells generally exit the cell cycle during G1 into the G0 phase.
The initiation of DNA replication, i.e. the transition from G1 to S, is a key step in the regulation of the cell division cycle. A plethora of intra-and extracellular signals is integrated during G1 phase of the cell cycle into a decision to withdraw from the division cycle, or to initiate S phase and hence to continue proliferation (Heichman and Roberts, 1994). Once S phase is initiated, control mechanisms ensure that all chromosomal DNA is replicated before chromosomes are segregated into the two daughter cells at mitosis (Nurse, 1994).
Cell fusion and nuclear transplantation experiments provided compelling evidence that quiescent cell nuclei are induced to initiate DNA replication when introduced into S phase cells (Graham et al., 1966; Harris et al., 1966; de Terra, 1967; Johnson and Harris, 1969). When synchronized cells were fused, S phase cells induced DNA replication only in G1 nuclei, but not in G2 nuclei (Harris et al., 1966; de Terra, 1967; Guttes and Guttes, 1968; Ord, 1969; Rao and Johnson, 1970). These results indicated that S phase cells contain dominant specific factors that trigger DNA replication and are evolutionarily conserved. Unreplicated G1 nuclei are the physiological substrates for the initiation of DNA replication, whilst re-replication in G2 nuclei is prevented until they have undergone mitosis (Romanowski and Madine, 1996).
The transition from G1 to S is regulated by a number of proteins within the cell, in particular the cyclins A, D and E, and their associated cyclin-dependent kinases (Cdks), particularly Cdk2.
One of the major mechanisms by which a replication-competent state during the G1 phase of the cell cycle is achieved involves the regulated assembly of pre-replicative complexes (pre-RCs) or xe2x80x9creplication licencesxe2x80x9d at origins of replication during G1 (Diffley et al., 1994, reviewed by Donovan and Diffley, 1996).
The pre-RC includes two heteromeric protein complexes, the minichromosome maintenance complex (MCM) and the origin recognition complex (ORC), together with the monomeric protein Cdc6 (reviewed by Dutta and Bell, 1997,; Newlon, 1997; Romanowski and Madine, 1997).
The six-subunit origin-recognition complex (ORC) binds specifically to S. cerevisiae autonomously replicating sequences (ARS) throughout the cell cycle (Bell and Stillman, 1992; Diffley and Cocker 1992; Aparicio et al.,1997; Liang and Stillman, 1997; Tanaka et al., 1997). Although origins of replication have been difficult to define in higher eukaryotes, homologues of the yeast ORC proteins have a similar function in that they are required for initiation of replication (Gavin et al., 1995; Carpenter et al., 1996; Coleman et al., 1996; Romanowski et al., 1996a; Rowles et al., 1996).
In yeast, it has been shown that the monomeric Cdc6 protein is essential for the initiation of DNA replication and is required for the assembly and maintenance of the pre-Rc (Kelly et al., 1993; Liang et al., 1995; Nishitani and Nurse, 1995; Piatti et al., 1995; Cocker et al., 1996; Muzi-Falconi et al., 1996, Detweiler and Li, 1997, 1998) but its role in mammalian cells is less well characterised (Yan et al., 1998).
The six members of the MCM protein family (MCM2-7) are also components of the pre-RC and association of these proteins with chromatin is required for initiation of DNA replication (Chong et is al., 1995; Dalton and Whitbread, 1995; Kubota et al., 1995; Madine et al., 1995a). During replication the MCM proteins become phosphorylated and displaced from chromatin (Kimura et al., 1994;Chong et al., 1995; Kubota et al., 1995; Madine et al., 1995a, 1995b; Todorov et al., 1995; Couxc3xa9 et al., 1996; Hendrickson et al., 1996; Krude et al., 1996). Cells arrested in vitro by serum starvation or contact inhibition lose chromatin-bound MCMs (after a few days). Although the total level of MCMs in the cells does not decrease greatly within 14 days, after 14 days it falls sharply. Cells which undergo differentiation in vitro (e.g. HL-60 cells induced to differentiate with DMSO or TPA) down-regulate MCM3 but not Orc2 (Musahl, Aussois Meeting on DNA Replication, Aussois, France, June 1997). Differentiated cells from tissues ex vivo do not express MCM proteins such as MCM2 and MCM5. In co-pending patent application GB 9722217.8, it is shown that MCM5 is absent from differentiated cells of the uterine cervix and breast.
The six MCM proteins MCM2-MCM7 form a multiprotein complex, which splits into two subcomplexes: MCM3 and MCM5 dimer; MCM2-4-6-7 tetramer. MCM3 and MCM5 may be displaced from chromatin during S phase more slowly than MCM2-4-6-7 (Kubota et al., 1997, EMBO J. 16, 3320-3331). MCMs are chromatin-bound in G1, displaced during S phase, and nuclear, although not bound to chromatin, in G2.
In yeast and Xenopus assembly of the pre-RC is sequential with ORC recruiting Cdc6, which results in recruitment of MCM proteins (Coleman et al, 1996).
Human Cdc6 amino acid sequence is disclosed in Williams et al., 1997, PNAS USA 94: 142-147, GenBank Acc. No. U77949 and in WO 97/41153.
Human MCM2 sequence is disclosed in Todorov et al., 1994, J. Cell Sci., 107, 253-265, GenBank Acc. No. X67334.
Human MCM3 sequence is disclosed in Thommes et al., 1992, Nucl. Acid Res., 20, 1069-1074, GenBank Acc. No. P25205.
Human MCM4 sequence is disclosed in Ishimi et al., 1996, J. Biol. Chem., 271, 24115-24122, GenBank Acc. No. X74794.
Human MCM5 sequence is disclosed in Hu et al., 1993, Nucleic Acids Res., 21, 5289-5293, GenBank Acc. No. X74795.
Human MCM6 sequence is disclosed in Holthoff et al., 1996, Genomics, 37, 131-134, GenBank Acc. No. X67334.
Human MCM7 sequence is disclosed in Hu et al., 1993, Nucleic Acids Res., 21, 5289-5293.
ATPase enzymatic activity has been reported for Cdc6 and for MCM proteins. (Zwerschke et al., 1994; Ishimi et al., 1997). These proteins may have other enzymatic activities, for instance helicase activity as reported by Ishimi et al., 1997.
Direct biochemical analysis of replication initiation in eukaryotic somatic cells has been impeded by the lack of an efficient mammalian cell-free DNA replication system to complement these cellular and genetic approaches.
We have previously developed a cell free system for initiating DNA replication under cell cycle control (Krude et al, 1997 and PCT/GB97/01751) in which there is provided:
(a) S phase cytosol or a fraction thereof in which are co-incubated
(b) G1 phase nuclei, and
(c) S phase nuclei or a fraction thereof and/or cyclins A and/or E complexed to their cognate cyclin dependent kinase (Cdk2).
Although our previous system allowed measurement of the initiation of DNA replication in vitro, preparations of G1 nuclei that are competent to replicate in that system are often contaminated with existing S phase nuclei. Hence we described various measures to identify these S phase contaminants such as BrdU labeling in vivo, and control incubations in G1 cytosol or dimethylaminopurine (DMAP). These measures work well for G1 nuclear populations with low levels of S phase contaminants, but are less satisfactory for preparations with high levels of S phase contaminants because the noise level rises relative to the initiation signal.
We have now refined this system and surprisingly found that by altering the method of preparation of G1 nuclei it is possible to dispense with the need for component (c) of the above system, and to improve the signal to noise ratio.
In particular, whereas we previously chemically induced is synchrony using a thymidine block and mitotic arrest by nocodazole, we found that the system may be improved using natural synchrony obtained by allowing untransformed cells to arrest growth in G0, and using such cells for the preparation of G1 nuclei.
In the examples which follow, we have used either mouse NIH 3T3 cells which show rigorous contact inhibition or human EJ30 cells which arrest in low serum. Release from contact inhibition is followed by entry into S-phase approximately 20 hours later for both NIH 3T3 and EJ30 cells, compared to about a 9 hour total G1 phase after release of HeLa cells from mitosis. We have used cells at various times after release from G0 as sources of G1 nuclei. This results in a high proportion of G1 nuclei that are competent to initiate in vitro as well as a lower frequency of S phase contaminants and hence a lower background noise. In addition, this protocol results in a simplified system that requires only competent G1 nuclei, S phase cytosol, deoxynucleoside triphosphates and an energy regenerating system to allow initiation of replication in vitro.
According to one aspect of the present invention there is provided a cell-free system for initiating DNA replication under cell cycle control, which system comprises:
a synchronous population of G1 nuclei which have been released from G0; and
S phase cytosol.
In a further aspect of the invention, there is provided a method for preparing G1 nuclei suitable for use in a cell-free system for initiating DNA synthesis which method comprises;
preparing a population of quiescent cells;
releasing said cells from quiescence;
cultivating said cells; and
harvesting the nuclei of said cells prior to the end of G1.
Furthermore the invention also extends to G1 nuclei obtainable by such a method and to the use of such nuclei in a cell-free DNA replication system, either of the invention or of the prior art.
Furthermore, the invention provides a method for conducting a cell-free DNA replication assay which comprises:
providing a synchronous population of G1 nuclei obtained from cells which have been released from G0; and
bringing said nuclei into contact with S phase cytosol under conditions suitable for DNA replication to occur.
Although not essential to the invention, the systems and assay may include one or more cyclin dependent kinases and/or their cognate cyclin components, including for example cyclin A and cyclin E both complexed to Cdk2, or a cyclin D complexed to Cdk 2, Cdk 4 or Cdk 6.
Furthermore, the present invention further provides the use of the cell free system disclosed above for identifying or obtaining an agent which modulates, e.g. inhibits or stimulates, DNA replication.
Such an agent may be identified by an assay method which comprises:
(a) treating a cell-free system according to the invention with a test substance; and
(b) determining DNA synthesis.
Substances identified by such a method may be modified to produce analogues with improved activity.
Substances including modified substances identified according to the invention may be formulated into a composition including at least one additional component, such as a pharmaceutically acceptable excipient.
Using the cell free DNA replication system of the invention the inventors have further investigated the molecular mechanisms following release from quiescence that establish a replication-competent state during G1. It is shown that quiescent cells lack Cdc6 and that MCM proteins in these cells are not associated with chromatin. It is shown that on release from quiescence, competence to replicate coincides with expression of Cdc6 and the binding of the MCM protein complex to chromatin. Furthermore it is shown that addition of Cdc6 to G1 phase nuclei accelerates G1 progression causing premature entry into S phase and increases the proportion of nuclei replicating, thus further enhancing the signal to noise ratio of the system. Furthermore, the results suggest that addition of MCM protein will increase MCM binding to chromatin and promote premature initiation of DNA replication.
Therefore according to a further aspect of the present invention, there is provided a cell-free system for initiating DNA replication under cell cycle control, which system comprises:
a synchronous population of G1 nuclei which have been released from G0;
S phase cytosol; and
a polypeptide supplied to the system;
wherein the said polypeptide is Cdc6 and/or at least one MCM protein.
In the context of the present application MCM protein is to be understood to refer to an MCM which might be or is selected from the group MCM2, MCM3, MCM4, MCM5, MCM6, MCM7. McM protein used may be mammalian such as human MCM protein or mouse MCM protein, amphibian such as Xenopus MCM protein or other eukaryotic such as Saccharomyces cerevisiae MCM protein and others as listed in Chong et al., 1996.
In the context of the present application, xe2x80x9cbeing suppliedxe2x80x9d does not mean that the the Cdc6 is necessarily obtained from other cell types but that the Cdc6 in the system is present at a higher concentration than would occur in the template nuclei at the time of isolation under natural conditions prior to initiation of DNA replication. All references to the polypeptide supplied should be construed accordingly.
In a further aspect of the invention, there is provided a method of bringing forward initiation of DNA replication under cell cycle control in a cell-free system by supplying Cdc6 and/or at least one MCM protein to the system.
In another aspect of the present invention, there is provided a method for preparing G1 nuclei suitable for use in a cell-free system for initiating DNA synthesis which method comprises;
preparing a population of quiescent cells;
releasing said cells from quiescence;
cultivating said cells;
harvesting the nuclei of said cells prior to the end of G1;
and bringing said nuclei into contact with Cdc6 and/or at least one MCM protein supplied to the system.
Furthermore the invention also extends to G1 nuclei obtainable by such a method and to the use of such nuclei in a cell-free DNA replication system, either of the present invention or of the prior art.
Furthermore, it provides a method for conducting a cell-free DNA replication assay which comprises:
providing a synchronous population of G1 nuclei obtained from cells which have been released from G0;
bringing said nuclei into contact with S phase cytosol and Cdc6 and/or at least one MCM protein supplied to the assay under conditions suitable for DNA replication to occur.
Furthermore, it provides a method for promoting binding of MCM to chromatin in G1 stage nuclei which comprises bringing said nuclei into contact with supplied Cdc6.
It is shown that the ability of the G1 phase nuclei to prematurely become competent to replicate is dependent on the permeable state of the nuclear envelope. Bringing forward of the nuclei to competence can only be achieved if the G1 phase nuclei are made permeable.
Therefore, in yet further embodiments, the invention extends to G1 nuclei, cell-free systems, methods and assays and their use in which the G1 nuclei are permeabilised.
The S phase cytosol used in the cell-free systems according to the invention is generally supplemented with nucleoside and deoxynucleoside triphosphates (NTPs and dNTPs respectively). Usually, one of the deoxynucleoside triphosphates will be labeled.